This project will elucidate the mechanisms of TRH biosynthesis and regulation in the mammalian hypothalamic tuberoinfundibular system, a hormone which has important effects in the neuroendocrine control of anterior pituitary TSH and prolactin secretion. The sequence of rat preproTRH will be determined by isolating immuno-positive clones from a rat Lambdagtll expression library selected by antibody probes specific for proTRH. Based on the nucleotide sequence of the cDNA encoding preproTRH an antisense mRNA probe will be prepared complementary to TRH mRNA, using the Riboprobe vector for in situ hybridization histochemistry. The hybridization probes will be used to determine the effect of electrolytic ablation of specific afferent projections to TRH-containing neurons in the paraventricular nucleus and hormonal feedback, cold exposure and suckling on TRH biosynthesis using quantitative computer imaging and analysis. The delineation of the afferents to TRH-containing neurons and their specific peptidergic and aminergic mediators will be accompanied by anterograde tracing methods using the lectin PHA-L, combined with immunocytochemistry at the light and electron microscopic levels.